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1.
Arterioscler Thromb Vasc Biol ; 33(12): 2792-9, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24072697

RESUMO

OBJECTIVE: Transforming growth factor-ß-activated kinase 1 (TAK1) is a mitogen-activated protein 3-kinase and an AMP-activated protein kinase (AMPK) kinase in some cell types. Although TAK1(-/-) mice display defects in developmental vasculogenesis, the role of TAK1 in endothelial cells has not been investigated in detail. APPROACH AND RESULTS: TAK1 downregulation (small interfering RNA) in human endothelial cells attenuated proliferation without inducing apoptosis and diminished endothelial cell migration, as well as tube formation. Cytokine- and vascular endothelial growth factor (VEGF)-induced endothelial cell sprouting in a modified spheroid assay were abrogated by TAK1 downregulation. Moreover, VEGF-induced endothelial sprouting was impaired in aortic rings from mice lacking TAK1 in endothelial cells (TAK(ΔEC)). TAK1 inhibition and downregulation also inhibited VEGF-stimulated phosphorylation of several kinases, including AMPK. Proteomic analyses revealed that superoxide dismutase 2 (SOD2) expression was reduced in TAK1-deficient endothelial cells, resulting in attenuated hydrogen peroxide production but increased mitochondrial superoxide production. Endothelial cell SOD2 expression was also attenuated by AMPK inhibition and in endothelial cells from AMPKα1(-/-) mice but was unaffected by inhibitors of c-Jun N-terminal kinase, p38, extracellular signal-regulated kinase 1/2, or phosphatidylinositol 3-kinase/Akt. Moreover, the impaired endothelial sprouting from TAK(ΔEC) aortic rings was abrogated in the presence of polyethylene glycol-SOD, and tube formation was normalized by the overexpression of SOD2. A similar rescue of angiogenesis was observed in polyethylene glycol-SOD-treated aortic rings from mice with endothelial cell-specific deletion of the AMPKα1. CONCLUSIONS: These results establish TAK1 as an AMPKα1 kinase that regulates vascular endothelial growth factor-induced and cytokine-induced angiogenesis by modulating SOD2 expression and the superoxide anion:hydrogen peroxide balance.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células Endoteliais/enzimologia , MAP Quinase Quinase Quinases/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Animais , Antioxidantes/farmacologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Peróxido de Hidrogênio/metabolismo , Interleucina-1beta/metabolismo , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/deficiência , MAP Quinase Quinase Quinases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Neovascularização Fisiológica , Oxirredução , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Fatores de Tempo , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
PLoS One ; 11(1): e0146645, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26751588

RESUMO

Epigenetic marks critically control gene expression and thus the cellular activity state. The functions of many epigenetic modifiers in the vascular system have not yet been studied. We screened for histone modifiers in endothelial cells and observed a fairly high expression of the histone plant homeodomain finger protein 8 (PHF8). Given its high expression, we hypothesize that this histone demethylase is important for endothelial cell function. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. As a functional readout endothelial migration and tube formation was studied. PHF8 siRNA attenuated the capacity for migration and developing of capillary-like structures. Given the impact of PHF8 on cell cycle genes, endothelial E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4 but not E2F1 expression in endothelial cells. Consistently, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Conclusion: PHF8 by controlling E2F4 expression maintains endothelial function.


Assuntos
Movimento Celular , Fator de Transcrição E2F4/metabolismo , Células Endoteliais/citologia , Histona Desmetilases/metabolismo , Fatores de Transcrição/metabolismo , Apoptose , Catálise , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Metilação de DNA , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Histonas/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Microcirculação , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Sítio de Iniciação de Transcrição
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